1) Field of the Invention
The present invention relates to methods for preparing extract products from the root of Polygonum multiflorum Thunb., to the extract products thus obtained, which have been demonstrated to have biological activities in enhancing liver cell regeneration and bone marrow stem cell proliferation, and to the uses of such extract products in the manufacture of medicaments for promoting cell proliferation, growth and/or differentiation.
2) Description of the Related Art
Stem cell refers to those cells capable of self-renewal and differentiation. Stem cells are most numerous during the embryonic period, and gradually decrease in number with aging. Thus, it was speculated that there is an important correlation/association between stem cells and aging. The stem cells in adults can generate a specific response toward the message in the environment, and generate new stem cells or differentiate into specific cells. When the stem cells receive a differentiation message, the stem cells rapidly reproduce in large amounts, and then finally proceed to differentiation. These stem cells are used for maintaining the balance of cells in adults, and replenish the number of cells that die due to natural causes or injuries.
The stem cells in bone marrow are divided into two types: (1) the hematopoietic stem cells which produce two more specialized types of stem cells, including lymphoid progenitor cells (which give rise to T and B lymphocytes) and myeloid progenitor cells (which give rise to leukocytes, erythrocytes, and megakaryocytes); and (2) the stromal cells which are the source of the cells making up the supporting structure in the bone marrow. The stromal cells have the characteristic of adhering to the bottom of plastic culture plates during culturing, and can differentiate into osteoblasts, chondrocytes, adipocytes, and even myoblasts. Stromal cells are required for the growth and differentiation of hematopoietic stem cells.
The production and number of stem cells will be extensively reduced as aging occurs, leading to various problems of aging, in which osteoporosis is the most common. The causes of osteoporosis include the loss of balance between bone formation and resorption. The osteoblastic cells derived from the osteoprogenitor cells are responsible for bone formation. Osteoprogenitor cells come from the stromal cells in the bone marrow. Dexamethasone and ascorbic acid can promote the proliferation growth of osteoprogenitor cells, and enable the cells to differentiate into matured osteoblasts. During the differentiating process, different markers of osteoblasts are expressed: There is the deposition of collagenous matrix first, and after 10 to 14 days, alkaline phosphatase (AP) is expressed.
Alkaline phosphatase is widely used as a biochemical marker for identification of osteoblast activity. While its actual function is unknown, it is currently believed that it participates in the skeletal mineralization process. After continuous culture to 21 days, the cells will secrete osteocalcein, and finally mineralize to form bone nodules.
It was known that the proliferation and differentiation of the stem cells would be affected by the growth factors, such as epidermal growth factor (EGF), granulocyte-monocyte colony stimulating factor (GM-CSF), etc. When the growth factors in the culture environment are changed, the stem cells differentiate into different cells along with the specificity of the growth factors. For example, GM-CSF can act on a specific receptor complex present on hematopoietic progenitor cells, and thus, can promote the proliferation and differentiation of the hematopoietic progenitor cells in the bone marrow into monocyte, neutrophil, etc. Therefore, GM-CSF may be used to treat the diseases related to leukocyte deficiency.
The recognition that somatic stem cells can be isolated and are able to renew a particular tissue motivated immediate efforts to apply these cells in the clinic. Bone marrow transplantation, albeit not successful in all circumstances, has become a mainstay in the treatment of hematological and some nonhematological diseases and cancers (Treleaven, J., and Barrett, J. 1992. Introduction to bone marrow transplantation. In Bone marrow transplantation in practice. J. Treleaven and J. Barrett, editors. Churchill Livingstone. New York, N.Y., USA. 3-9). Extensive skin lesions are now being treated with the use of autologous and even nonautologous grafts generated by the ex vivo expansion of epidermal cells (Green, H. (1989), Lab. Invest. 60:583-584). The reconstruction of damaged articular cartilage has been attempted using ex vivo expanded chondrogenic cells (Brittberg, M. et al. (1994), N. Engl. J. Med. 331:889-895). More recently, it has also been suggested that skeletal tissue, muscle, and even nervous tissue can be regenerated from stem cell populations. Potential applications extend beyond tissue regeneration, into the realm of gene transfer and gene therapy.
With the advance of molecular techniques, it is envisioned that stem cells could be engineered to replace or repair a defective gene. Because of their self-renewal and ability to regenerate a tissue, transgenic stem cells could provide a long-lasting clinical benefit to a recipient. Although the precise techniques for accomplishing these goals are not yet in hand, our biotechnological imaginations have run wild with the hope of recreating organs, correcting genetic diseases, and improving the quality of life as we age. The realization that cells with extensive potential for growth and differentiation occur in a variety of tissues also provides novel angles for understanding disease mechanisms. Since stem cells regulate the dynamics of normal tissues, a surprising range of disorders, including gastric atrophy, Alzheimer's disease, and, perhaps more intuitively, various forms of cancer, can be traced to altered stem cell function.
The trend toward defining stem cells primarily based on our ability to manipulate them in culture should also inspire us to devise novel models of these diseases, by analyzing genetically altered or carcinogen-treated stem cells either in vitro or in vivo after transplantation into host animals. Thus, even without improved tissue engineering or replacement, the study of stem cells may deepen our understanding of their pathogenic roles and facilitate the design of novel treatments.
The major causes of liver cirrhosis include chronic alcoholism, viral infection and metabolic lesion. Currently, there is no therapy that is effective in the treatment of liver cirrhosis. Recently, it is reported in literature that hepatocyte growth factor may be used in the treatment of human liver fibrosis and chronic liver failure (Matsuda Y. et al. (1995), Journal of biochemistry, 118 (3): 643-9; Ueki T et al. (1999), Nature Medicine, 5 (2): 226-30).
Mammalian hepatocytes have been used in the investigation of cell growth and differentiation mechanism for a long time. However, shortly after isolation, mammalian hepatocytes will lose their characteristics and their growth ability will be limited. Many researchers have endeavored to develop a methodology for the growth of normal hepatocytes, but no successful results have been reported.
Fleeceflower root is the dried root tuber of Polygonum multiflorum Thunb. (Family Polygonaceae), and it has been used as a traditional Chinese medicine called Heshouwu (Latin Title: Radix Polygoni Multiflori) for a long time.
Fleeceflower root is produced in most parts of China, in Taiwan and in Japan. It is collected in autumn and winter when leaves wither. After cutting off the two ends thereof, the collected fleeceflower root is washed clean, cut into pieces, and then dried. Fleeceflower root tastes sweet, astringent and bitter in flavor, slightly warm in nature, and it has affinity to the liver and kidney channels.
Fleeceflower root may be used in processed or unprocessed form. The processed fleeceflower root is slightly warm but not dry and not greasy. It functions in replenishing the liver and kidney, benefiting essence and blood, and astringing primordial energy. This herb is mild in action and very effective in tonification. It is said that anyone who takes this herb regularly for a long period can prolong his life. It serves to treat insufficiency of both the liver and kidney, deficiency of essence and blood, and disability of the lower-jiao. The unprocessed fleeceflower root can also function in clearing away toxins and lubricating the bowels, serving to treat scrofula, carbuncle and constipation due to dryness of the bowels.
Fleeceflower root is known to contain the following chemical components: emodin, chrysophanol, physcion, rhein, chrysophanol anthrone, resveratrol, piceid, 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucopyranoside, 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucopyranoside-2″-O-monogalloyl ester, 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucopyranoside-3″-O-monogalloyl ester, gallic acid, catechin, epicatechin, 3-O-galloyl(−)-catechin, 3-O-galloyl(−)-epicatechin, 3-O-galloyl-procyanidin B-2, 3,3′-di-O-galloyl-procyanidin B-2, and β-sitosterol.
Fleeceflower root may be used in the treatment of hyperlipemia, lymphadenitis, carbuncles, urticaria with itching, and constipation.
It is reported in literature that hydroxyl anthraquinone derivatives contained in Polygonum multiform roots have the vasorelaxant effect (Huang, H. C. et al., European Journal of Pharmacology 198:211-3, 1993) and the effect of reducing the incidence of myocardial ischemia-reperfusion injury (Yim, T. K. et al., Phytotherapy research 14:195-99, 2000). It is also reported in literature that stilbene glucosides contained in Polygonum multiform roots have the blood lipid-lowering effect (Kimura Y, Planta Medica 49:51-54, 1983) and the antioxidation effect (Chen, Y. et al., Journal of Agricultural and Food Chemistry 47:2226-8, 1999).
WO 95/30427 disclosed the use of extracts of the Chinese herb Polygonum multiflorum in the treatment of hyperglycemia. According to the disclosure of WO9530427A1, the root of Polygonum multiflorum was extracted with 0.1 N NH4OH (20:1) (w/v) and centrifuged (1,000×g). The resultant supernatant was then applied to a Sephadex G-25 column and eluted with distilled deionized water. Three fractions were collected and demonstrated to exhibit a high insulin potentiating activity in fat cell assays and to have the effect of lowering blood sugar levels.
U.S. Pat. No. 6,200,569 disclosed and claimed a method for decreasing the glycosylated hemoglobin level or blood glucose level in a hyperglycemic patient, in which a water extract or a dilute acidic extract of Polygonum multiform roots, or Cinnamomum bark, or a mixture thereof, was administered to the patient. According to Example 2 of U.S. Pat. No. 6,200,569, the roots of Polygonum multiform were cut up and ground into small pieces, followed by extraction with water. The obtained water extract was then assayed for insulin potentiating activity.
CN 1306837A disclosed the use of Polygonum multiform roots and the extracts thereof in preventing and treating osteoporosis.
According to the Applicant's knowledge, none of the abovementioned patent publications and scientific articles has disclosed the production of methanol-extracted products of Polygonum multiform roots and the further extracted fractions thereof, as well as the biological effect(s) of these extracted products of Polygonum multiform roots upon bone marrow stem cells and liver cells.